Dyes employed in many production cycles are characterized by high toxicity and persistence in the environment, and conventional wastewater treatments often fail to reach high removal efficiencies. Consequently, there is an increasing research demand aimed at the development of more efficient and sustainable technologies. A two-step strategy consisting of dye sorption followed by sorbent bio-regeneration is proposed here, with a special focus on the regeneration step. The objective of this study was to establish the best operating conditions to achieve regeneration of dye-loaded polymers and concurrently the ultimate removal of the dyes. To this aim, the bio-regeneration of the Hytrel 8206 polymer, used as a sorbent material to remove Remazol Red dye from textile wastewater, was investigated in a two-phase partitioning bioreactor (TPPB) under alternated anaerobic–aerobic conditions. Comprehensive analysis of operational parameters, including sorbent load and initial contamination levels, was conducted to optimize bio-regeneration efficiency. Experimental data demonstrated high regeneration efficiencies (91–98%) with biodegradation efficiencies up to 89%. This study also examines the biodegradation process to investigate the fate of biodegradation intermediates; results confirmed the successful degradation of the dye without significant by-product accumulation. This research underscores the potential of TPPB-based bio-regeneration of polymeric sorbent material for sustainable wastewater treatment, offering a promising solution to the global challenge of dye pollution in water resources. [ABSTRACT FROM AUTHOR]